Journal of Tea Science

Determination of 7 Herbicide Residues in Tea Using Gas Chromatography - Tandem Mass

CHEN Hong-ping, LIU Xin, WANG Qing-hua, JIANG Ying

Journal of Tea Science. 2011, 31(4): 283-288. doi:10.13305/j.cnki.jts.2011.04.008

Abstract ( 609 )

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A gas chromatography-tandem mass spectrometric (GC-MS/MS) method has been developed for simultaneous determination of 7 herbicide residues in tea by accelerated solvent extraction (ASE) and gel permeation chromatography (GPC) cleaned up. The target analytes were extracted with acetonitrile using ASE,eluted with ethyl acetate/cyclohexane (1:1), cleaned up by GPC. The detection of the target analytes was used GC-MS/MS with multi-reaction-monitor (MRM) mode. At the spiked levels of 0.010μg/g and 0.100μg/g, the average recoveries were from 78.2% to 93.6%, and the relative standard deviations (RSDs) were from 5.8% to 19.1% (n=5). The limits of detection were 2.0~8.0ng/g. The method was simple, automated, reliable, accurate, sensitive and appropriate for the determination of 7 herbicide residues in tea.

Study on the Relationship between Virulence and Genetic Structure of Four Wild Isolates of Euproctis pseudoconspersa Nuclear Polyhedrosis Virus

FU Jian-yu, XI Yu, TANG Mei-jun, YIN Kun-shan, XIAO Qiang

Journal of Tea Science. 2011, 31(4): 289-294. doi:10.13305/j.cnki.jts.2011.04.009

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The virulence determination of four Euproctis pseudoconspersa nuclear polyhedrosis virus (EpNPV) isolates showed that Zhejiang isolate is the most toxic one (LC₅₀=2.50×10⁶PIB/mL), and Hunan isolate is the weakest isolate (LC₅₀=13.36×10⁶PIB/mL). The virulence of different isolates in order is Zhejiang (ZJ), Guizhou (GZ), Hubei (HB) and Hunan (HN), besides GZ and HB isolates have similar virulence, which LC₅₀were 9.50×10⁶PIB/mL and 7.31×10⁶PIB/mL respectively. Five genes of the four isolates about virus reproduction, interaction between virus and host and virulence were determined and sequenced, genetic structure based on these genes among isolates was analysed, the result indicated the similarity of HB and GZ, HN are highest (99.7%), and that of ZJ between HN isolate are lowest (99.4%). The NJ and UPGMA phylogenetic trees based on five genes also showed that HB and HN isolates first clustered into one group, then clustered with GZ, finally clustered with ZJ isolate, which indicated that the genetic distance among ZJ and other three isolates are relatively far. Both virulence determination and genetic structure analysis suggested virulence and heredity among four virus isolates are closely related.

Isolation and Identification of highly pathogenic Beauveria bassiana against Thrips

WANG Jun-ping, ZHENG Chang-ying

Journal of Tea Science. 2011, 31(4): 295-299. doi:10.13305/j.cnki.jts.2011.04.010

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12 different entomopathogenic fungi isolated from diseased Drosicha corpulenta Kuwana showed high virulence with 70%~90% mortality. CYT10 strain was identified based on the morphological and molecular characteristics. Conidiogenous cells were short-globose to flask-shaped and the conidiophore elongated in a long zig-zag extension after each conidium is produced. The result of Blast search indicated that the ITS and EF1-α sequence of CYT10 showed a high similarity with those of other strain of Beauveria bassiana (>95%). Phylogenetic analysis showed that it was closely related to ARSEF-1185 strain, which was belonging to B. bassiana. CYT10 strain was classified as a member of the species B. bassiana based on the morphological characteristics and analysis of DNA sequences.

Research on Tea Liquor Variation in Terms of Light Absorption Value during Black Tea Fermentation Process

LIU Xiao-dong, LIU Yu-fang, GAN Chun-ping, YANG Chun, TANG Zhou-bin

Journal of Tea Science. 2011, 31(4): 300-304. doi:10.13305/j.cnki.jts.2011.04.003

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In order to find out the color variation of the theaflavin aqueous solution in the tea liquor, during the fermentation of the black tea, this experiment measures the light absorption value of tea liquor manufactured with different tea cultivars, fresh leaves with different tenderness and processed in different seasons and different fermentation time by using the spectrophotometer at 460nm wavelength. Results of the experiment showed that the light absorption value and its maximum value and the velocity of variation are different under the above conditions. However, the light absorption value of the tea liquor changed as low - high -low with the extension of fermentation time, which is in accordance with the variation of the theaflavin content during the black tea fermentation.

Study on the Quality Evaluation and Product Discriminate of Oolong Tea

SUN Wei-jiang, DONG Qing-hua, ZHOU Wei-long, DONG Xiu-yun

Journal of Tea Science. 2011, 31(4): 305-312. doi:10.13305/j.cnki.jts.2011.04.004

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The model of quality evaluation and products discrimination of Oolong tea based on chemical composition, factor and discriminant analysis were studied with Guangdong, southern and northern Fujian Oolong tea as the samples. Seven factors with cumulative variance of 85.041% were obtained along with factor analysis, set the factors as the independent variable, and discriminating classification of the samples area,variety and grade use discriminant analysis. Then the discrimination functions were established. Results of cross-validation and resubstitution of testing samples demonstrated the correct rates of discrimination model of area and variety was satisfactory. Recognition accuracy of the resubstitution and cross-validation of area were 97.6% and 94.0%, which of variety was 86.9% and 75.0%. Seven score function of quality factor and one comprehensive evaluation function were established for Oolong tea, and three discrimination functions for producing area and six of variety were obtained and the discrimination results were satisfactory.

Study on Optimized Technical Parameters of the Liquid Fermented Green Tea Wine

LI Jian-fang, ZHOU Ying, ZHOU Feng, WANG Rong-rong, FANG Ling

Journal of Tea Science. 2011, 31(4): 313-318. doi:10.13305/j.cnki.jts.2011.04.005

Abstract ( 455 )

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Taking Xinyang Maojian tea as material, the production technology of tea wine was studied. Single factor experiments of the effects of inoculation number, sugar content, pH and temperature on fermentation were carried out. The optimum fermentation conditions of tea wine were determined by orthogonal experiments as follows: inoculum quantity 0.3‰, sugar content 180g/L, pH4.6, fermentation temperature 24℃ and fermentation time 12d. The product was characterized in special flavor, high nutritional components and health function.

Analysis of Codon Usage in Tea Plant (Camellia sinensis)

ZHAO Yang, YANG Yang, LIU Zhen, YANG Pei-di

Journal of Tea Science. 2011, 31(4): 319-325. doi:10.13305/j.cnki.jts.2011.04.013

Abstract ( 478 )

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The coding sequence of 134 selected proteins in tea plant (Camellia sinensis) were analyzed by CodonW and CUSP program in this investigation, and the frequency of codon usage which encoded amino acids was calculated and compared with four classes of representative organisms including human (Homo sapiens), yeast (Saccharomyces cerevisiae), Escherichia coli. Bacteria and fruit fly (Drosophila melanogaster). These results showed that codon preference of tea plant was differed in various degrees with these model organisms. Accomparison of codon usage frequency of tea plant with dicotyledons and monocotyledons plants including Arabidopsis thaliana, soybean (Glycine max), cotton (Gossypium hirsutum), rice (Oryza sativa), wheat (Triticum aestivum), the consequence indicated that codon usage frequency of tea plant was obviously differed from these monocotyledons such as Oryza sativa and Triticum aestivum, but had a consistency in codon preference with these dicotyledons such as Arabidopsis thaliana, Glycine max and Gossypium hirsutum. These results showed an important guidance for tea plant gene-transforming and the selection of the high-efficient expression system.

Expression and Optimization of Anthocyanin Reductase Gene of Tea Plant [Camellia sinensis (L.) O. Kuntze] in Escherichia coli

LUO Yang, WANG Hong-xue, WANG Yun-sheng, LU Zhong-wei, LIU Ya-jun, SHAN Yu, CHEN Xiao-tian, YE Hui, GAO Li-ping, XIA Tao

Journal of Tea Science. 2011, 31(4): 326-332. doi:10.13305/j.cnki.jts.2011.04.014

Abstract ( 577 )

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Anthocyanin reductase is a key enzyme in the biosynthesis of EC and EGC in tea plant. The open reading frame of anthocyanin reductase gene (ANR), which encoding a 337 amino acids protein, was cloned from tea plant 〔Camellia sinensis (L.) O. Kuntze 〕 by RT-PCR. The deduced protein molecular weight was 37kD and its theoretical isoelectric point was 6.54. The gene was cloned into the expression vector pET32a (+) for expression in prokaryotic cells. The SDS-PAGE results showed that the anthocyanin reductase peoteins was expressed in Escherichia coli rosetta. The optimal inducing conditions including time, temperature, IPTG concentration, ampicillin concentration were studied. The deduced protein was purified and its activity was detected by HPLC.

Study on Expression Conditions of EGCG-O-Methyltransferase in Recombinant Escherichia coli Bacteria

FEI Dong-mei, LIN Zhi, LU Hai-peng, ZHANG Yue, TAN Jun-feng, GUO Li

Journal of Tea Science. 2011, 31(4): 333-340. doi:10.13305/j.cnki.jts.2011.04.001

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EGCG3"Me could be produced from EGCG catalyzed by EGCG-O-Methyltransferase. Taken the yield of EGCG3"Me as main index, the present study focused on the producing conditions of EGCG-O-Methyltransferase induced by IPTG in recombinant E. coli bacteria. Results showed that the optimum producing conditions were as follows: the concentration of IPTG was 0.05mmol/L, the induction time was 20h, the initial pH of medium was 7.0 and the induction temperature was 20℃.

Effects of Enzymatic Treatment on Aroma Quality of Anxi Tie-Guan-Yin Harvested in Summer

XIAO Shi-qing, ZHANG Xue-bo, DU Xian-feng, LIN Duan-lian

Journal of Tea Science. 2011, 31(4): 341-348. doi:10.13305/j.cnki.jts.2011.04.006

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In order to improve the quality of Anxi Tie-Guan-Yin harvested in summer, laccase, α-galactosidase and combined-enzyme were applied in the processing of Anxi Tie-Guan-Yin. The quality of the tea samples were compared by sensory evaluation and the volatile constituents were investigated by gas chromatograph/mass spectrometer (GC-MS). The results showed that the relative level of the total aroma oil in the tea samples treated by enzymes were more than that in the untreated samples. α-Galactosidase treatment greatly improved the contents of aldehydes and alcohols. Laccase treatment greatly improved the contents of esters. Both of the two enzymes significantly increased the contents of alkenes. Nerolidol, indole and farnesene were found to be the most important odor-active compounds in Tie-Guan-Yin. The relative contents of these three compounds were all increased in tea samples by different enzymatic treatments. The sensory evaluation showed that the grassy smell of tea reduced and the flowery smell appeared by combined-enzyme treatment. The results indicated that enzymatic treatment improved the aroma quality of Anxi Tie-Guan-Yin harvested in summer.

Status of the Total Amount of Rare Earth Oxides in Tea and Its Leaching Characteristics

SHI Yuan-zhi, HAN Wen-yan, MA Li-feng, FANG Li

Journal of Tea Science. 2011, 31(4): 349-354. doi:10.13305/j.cnki.jts.2011.04.011

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In order to figure out the status of total amount of REE oxides（∑REOs） in tea and its leaching characteristics, the statues of ∑REOs in 1245 tea samples from 2007 to 2010 were statistical analyzed, and the leaching characteristics on the different ∑REOs with different processing technology were studied. The results showed that the Median of the ∑REOs in Chinese tea was about 2mg/kg, and the ranges were from ND to 13.80mg/kg, about over 90% of tea were under 5mg/kg. The ∑REOs in over half of Oolong teas were more than 2mg/kg, but for the other tea such as green tea, scented tea, less than 30% of those teas were more than 2mg/kg. When the tea was grinded, the leaching rate of the ∑REOs was increased significantly than the original tea. And the processing technology maybe was the major factor to impact on the leaching rate of the ∑REOs. There were some rules about the leaching rate followed by low to high: needle-shaped tea< flat-shaped tea (such as Longjin tea)< Curl-shaped tea < powdered tea. The ∑REOs was highest in the first brew, and then was decreased along with the 2^nd and the 3^rd brews. The Powdered Oolong tea has the highest leaching rate about the ∑REOs, the leaching rate was about 60%, but even the ∑REOs in the powdered Oolong tea has reached to 6mg/kg,the ∑REOs in the brew still under the limited value about the standard in drinking water. The needle-shaped tea has the lowest leaching rate about the

Establishment of the Predictive Models for Quantitative Analysis of Major Components in Green Tea Infusion by Near Infrared Spectroscopy (NIRS)

WANG Yu-xia, XU Rong-rong, REN Guang-xin, WANG Sheng-peng, WAN Xiao-chun, ZHANG Zheng-zhu

Journal of Tea Science. 2011, 31(4): 355-361. doi:10.13305/j.cnki.jts.2011.04.002

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Based on the large amounts of analyzing of major components in samples of green tea infusion, combining the chemical analysis results with near infrared spectra of tea infusion samples scanned by near infrared spectroscopy, this paper established and optimized quantitative analysis models of water extract, tea polyphenols, free amino acids, soluble sugars, caffeine, total catechins, EGCG, EC, EGC, EGC and GA by using partial least squares (PLS) method. Each of the 11 predictive models was applied for the external inspection. Results showed that almost all the decision coefficients of cross test and predictive models were exceed 95%, except for EGC and GA. RPDs of models were exceed 3, except for GA with a RPD of 2.67. The established models present high stability and predictive accuracy. This paper provided a new method for the analysis of major quality components in tea infusion and tea beverages by near infrared spectroscopy.

Flows and Mass Balance of Heavy Metals in Typical Tea Ecological Systems in Zhejiang Province, China

MA Wan-zhu, ZHANG Ming-kui

Journal of Tea Science. 2011, 31(4): 362-370. doi:10.13305/j.cnki.jts.2011.04.012

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Four tea production sites, located in Changxing, Lanxi, Shaoxing and Longquan, were selected to study the flows of Zn, Cu, Pb, Cd, Hg, and As loaded by organic manure, chemical fertilizers, atmospheric deposition, tea harvest, and surface runoff in tea production system through one-year field monitoring. Annual mass balance of the heavy metals in the tea production system and its influencing factors were also discussed. The results showed that the flows of the heavy metals in input materials and agricultural products and drainages in the tea production systems varied spatially with investigated samples and fields. Inputs of Zn, Cu, and Pb in the tea gardens were mainly from atmospheric deposition, followed by organic manure. That of As was mainly from atmospheric deposition, followed by chemical fertilizers. Those of Cd and Hg were mainly from chemical fertilizers. In the tea gardens where organic manures were applied as main nutrients, annual input amounts of Zn, Cu, Pb, Cd, Hg, and As were 1026.4, 366.8, 77.0, 3.14, 0.83, and 40.3g/hm², respectively. While annual output amounts of Zn, Cu, Pb, Cd, Hg, and As were 187.1, 63.2, 10.7, 0.55, 0.36, and 32.9g/hm², respectively. Inputs of Zn, Cu, Cd and Hg in the tea garden were mainly from organic manure, followed by atmospheric deposition. Those of Pb and As were mainly from atmospheric deposition, followed by organic manure. Outputs of Zn and Cu in the way of surface runoff were higher than those in the way of tea harvest, and those of Pb, Cd, Hg, and As in the way of tea harvest were much higher than those in the way of surface runoff. Annual mass balances of Zn, Cu, Pb, Cd, Hg, and As were 313.0, 11.8, 41.2, 2.95, 0.22, and 2.18g/hm²for tea gardens where chemical fertilizers were applied as main nutrients, and 849.3, 303.4, 66.3, 2.59, 0.47, and 7.43g/hm²for tea gardens where organic manures were applied as main nutrients. The annual mass balances of the heavy metals were higher in the tea gardens where organic manures were applied as main nutrients than those where chemical fertilizers were applied as main nutrients.

Isolation and Identification of Thermophilic Fungi during the Fermentation of Puer Tea

YANG Rui-juan, LU Jie, YAN Liang, YANG Liu-xia, LI Chen-chen, JIANG Shu, SHENG Jun

Journal of Tea Science. 2011, 31(4): 371-378. doi:10.13305/j.cnki.jts.2011.04.007

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Thermophilic fungi were isolated and identificated by the method of sequencing during traditional fermentation of puer tea. Sampling were carried out every day. Many types of microorganisms including thermophilic fungi were purified and Identified. Isolated thermophilic fungi were cultured at different temperatures and different pH, the growth state of thermophilic microorganisms were observed. This article showed a comprehensive analysis of microorganisms during the fermentation of puer tea, and identified the isolated microorganisms using molecular biology method. Thermophilic microorganisms which play an important role during the fermentation were discovered, it laid the foundation to reveal the mechanism of Puer tea fermentation at high temperature.

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